We plan to continue the following studies: Enzymology (and mechanism) of mtDNA Replication: (A) Nicking-closing Enzyme - We have detected this enzyme in rat liver mitochondria and plan to continue its purification and the study of its properties and its role in mtDNA replication. (B) mtDNA Gyrase - Results with novobiocin on isolated mitochondria suggested the presence of a DNA gyrase; we are continuing to try to isolate this enzyme and then to study its properties and role. (C) mtDNA Polymerase - We plan to complete the purification of this enzyme, to study its relationship to gamma polymerase, and to use it in studies on mtDNA replication. (D) Accessary Replication Proteins -Other proteins or enzymes which may plan accessary rolls to mtDNA polymerase will be sought. (E) D-loop DNA - Does mtDNA replication "hesitate" at the D-loop stage because of properties of superhelices or because of some special (e.g., protein binding) nucleotide sequence? This problem will be studied using a replication system comprising mtDNA polymerase and either mt nicking-closing enzyme or gyrase, and by sequencing the D-loop region. Intraspecific mtDNA Polymorphism - Using restriction endonucleases, we have found two types of mtDNA in a single species of rat (a single rat possessing one type). We plan to: (1) study the differences in structure, including sequence, bewteen the two types; (2) seek possible phenotypic differences; (3) perform experiments, heretofore impossible or inconclusive, based on the distinguishability of the two intraspecific mtDNAs; e.g., (a) prove the existence of maternal inheritance in mammals, (b) elucidate the basic mechanism, passive dilution or active suppression, which results in paternal mtDNA not being inherited; (c) determine whether paternal DNA injected into the animal finds its way into the mitochondrion and is replicated there. Dense DNA - What is the composition, structure, origin and role of a new mtDNA containing complex of buoyant density 1.76 we have isolated? The complex resembling the E. coli chromosome, consists of many loops emanating from a hazy core which contains electron opaque regions.